Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: The detection of three independent benzimidazole resistance mutations in a single Ancylostoma caninum population from a Golden Retriever breeding kennel in Sao Paulo, Brazil

doi: 10.1016/j.ijpddr.2026.100643

Figure Lengend Snippet: Illumina sequencing of amplicons from the isotype-1 β-tubulin gene encompassing codons 134, 167, 198 and 200. Panel A: Schematic representation of the A. caninum isotype-1 β-tubulin gene model and positions of the two amplified fragments; “exon 4, codons 134/167 fragment” and “exon 5, codons 198/200 fragment”. The vertical red bars on exons 4 and 5 indicate the positions of codons 134, 167, 198 and 200 respectively. Panel B: “Exon 4, codons 134/167 fragment” amplicon deep Illumina sequencing data. An amplicon was successfully generated from the TNA re-extraction 1 and TNA re-extraction 2 preparations in 6/10 replicate PCR reactions (indicated by the orange dots at the base of the chart) and 4/10 replicate PCR reactions (indicated by the blue dots at the base of the chart) respectively. Each of these amplicons was deep sequenced and the chart shows the relative proportions of the ASVs generated for each amplicon expressed as percentages (Y-axis). ASVs carrying the Q134H(CA A > CA T ) SNP are indicated in blue, ASVs carrying the F167Y(T T C > T A C) SNP are indicated in yellow and the remaining ASVs with susceptible genotypes at these two codons are indicated in grey. The table on the right shows the total number of reads mapping to the reference A. caninum isotype-1 β-tubulin sequence ( DQ459214.1 ) for each amplicon ordered as in the chart and percentage of the reads corresponding to the ASV carrying either the Q134H(CA A > CA T ) or F167Y(T T C > T A C) SNPs. Panel C: “Exon 5, codons198/200 fragment” amplicon deep Illumina sequencing data. An amplicon was successfully generated from the TNA re-extraction 1 and TNA re-extraction 2 preparations in 5/10 replicate PCR reactions (indicated by the orange dots at the base of the chart) and 7/10 replicate PCR reactions (indicated by the blue dots at the base of the chart) respectively. Each of these amplicons was deep sequenced and the chart shows the relative proportions of the ASVs generated for each amplicon expressed as percentages (Y-axis). ASVs carrying the F200Y(T T C > T A C) SNP are indicated in red and the remaining ASVs with susceptible genotypes at codons 198 and 200 are indicated in grey. The table on the right shows the total number of reads mapping to the reference A. caninum isotype-1 β-tubulin sequence ( DQ459314.1 ) for each amplicon ordered as in the chart and percentage of the reads corresponding to the ASV carrying the F200Y(T T C > T A C) SNP.

Article Snippet: A positive control TNA sample was made for the Oxford Nanopore Technologies (ONT) amplicon sequencing by pooling equal amounts of TNA preparations made from 12 different hookworm positive canine fecal samples from Orlando, Florida to Antech.

Techniques: Illumina Sequencing, Amplification, Generated, Extraction, Sequencing

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: The detection of three independent benzimidazole resistance mutations in a single Ancylostoma caninum population from a Golden Retriever breeding kennel in Sao Paulo, Brazil

doi: 10.1016/j.ijpddr.2026.100643

Figure Lengend Snippet: Oxford Nanopore amplicon sequencing of the near-full length A. caninum isotype-1 β-tubulin gene Panel A: Schematic representation of the A. caninum isotype-1 β-tubulin gene model. The vertical red bars on exons 4 and 5 indicate the positions of codons 134, 167, 198 and 200 respectively Panel B: An amplicon corresponding to the near-full length A. caninum isotype-1 β-tubulin gene was successfully generated from the TNA re-extraction 1 and TNA re-extraction 2 preparations in 7/10 replicate PCR reactions (indicated by the orange dots at the base of the chart) and 6/10 replicate PCR reactions (indicated by the blue dots at the base of the chart) respectively. Each of these amplicons was deep sequenced using Oxford Nanopore Technologies (ONT) sequencing. Since the error rate of ONT sequencing is too high to fulfill the minimum requirements for the DADA2 model, it was not possible to generate ASVs for the 3547 bp amplicon. Consequently, SNPs were detected at each nucleotide position, relative to the A. caninum isotype-1 β-tubulin reference sequence ( DQ459314.1 ) using Minimap2 (2.28-r1209) ( , ) and their frequencies calculated with Bam-readcount (0.8) . The histogram chart shows the frequencies all the non-synonymous SNPs detected in the A. caninum isotype-1 β-tubulin gene in each of the replicate PCRs. The table on the right shows the total number of reads mapping to the reference A. caninum isotype-1 β-tubulin sequence and the frequencies of each of the non-synonymous SNPs ordered as in the chart. Panel C: An amplicon corresponding to the near-full length A. caninum isotype-1 β-tubulin gene was generated from a positive control sample. This single positive control pooled sample was made from individual TNA samples prepared from 12 different hookworm positive canine fecal samples from Orlando, Florida prepared within 1 week of fecal collection. The amplicon was successfully generated from 3/3 replicate PCR and the Q134H(CA A > CA T ) or F167Y(T T C > T A C) SNPs detected at similar frequencies in each case.

Article Snippet: A positive control TNA sample was made for the Oxford Nanopore Technologies (ONT) amplicon sequencing by pooling equal amounts of TNA preparations made from 12 different hookworm positive canine fecal samples from Orlando, Florida to Antech.

Techniques: Amplification, Sequencing, Generated, Extraction, Positive Control

Journal: International Journal for Parasitology: Drugs and Drug Resistance

Article Title: The detection of three independent benzimidazole resistance mutations in a single Ancylostoma caninum population from a Golden Retriever breeding kennel in Sao Paulo, Brazil

doi: 10.1016/j.ijpddr.2026.100643

Figure Lengend Snippet: Haplotypic relationship of the 134H(CA T ), 167Y(T A C) and 200Y(T A C) benzimidazole resistance SNPs as determined by ONT sequencing of the near full-length isotype-1 β-tubulin gene. The ONT sequence reads from the TNA re-extraction 1 and 2 preparations that aligned to the A. caninum isotype-1 β-tubulin reference sequence ( DQ459314.1 ) were converted to multiple sequence aligned FASTA format with gofasta (1.2.1) . Each read was then annotated with its genotype at codons 134, 167, 198, and 200 with an R script using tidyverse (1.1.5) and Biostrings (2.72.0) packages and reads with the same resistance genotype at the multiple codon positions were collapsed into a single haplotype. The bar shows the relative proportions of ONT sequence reads (as percentages) with the different codon 134, 167,198 and 200 genotypes. The resistance genotypes are shown in red, yellow and blue text for codons 200, 167 and 134 respectively and susceptible genotypes shown in black text.

Article Snippet: A positive control TNA sample was made for the Oxford Nanopore Technologies (ONT) amplicon sequencing by pooling equal amounts of TNA preparations made from 12 different hookworm positive canine fecal samples from Orlando, Florida to Antech.

Techniques: Sequencing, Extraction